Project Summary/Abstract Single-cell technologies have contributed to the understanding of underlying order in heterogenous samples, providing a platform to identify rare cell types with implications to human disease. These platforms are largely based on analyses of single-cell suspensions and do not provide a spatial context to the findings. It is becoming increasingly evident that the tissue architecture is a critical parameter and contributes significantly to the overall biological state of a sample. High-parameter imaging technologies enable the identification and localization of rare cell types in heterogenous tissue samples. Currently available high-parameter imaging technologies are time-consuming, limited in the number of parameters that can be reproducibly imaged and/or are cost-prohibitive for broad use in the scientific community. Akoya Biosciences Inc. has exclusively licensed a new high-parameter imaging technology termed CODEX (CO-Detection by indEXing) that is robust, inexpensive and can quickly acquire fluorescence data across tens of parameters. The CODEX platform consists of a set of specialized biologic reagents, including tagged antibody and oligo products, as well as a fluidics robot that can be attached to any inverted microscope. The broad applicability of this technology will enable any lab, ranging from small academic labs to large pharmaceutical research entities, to acquire high-parameter imaging data. These analyses will enable a deep understanding of disease mechanisms, therapeutic targets and overall tissue architecture. The goal of Akoya Biosciences Inc. is to commercialize this technology through the production of the fluidics robot as well as reagent kits for antibody staining and signal rendering. The current version of the technology was developed for the analysis of fresh-frozen (FF) tissue samples; however, most tissue samples are stored as formalin-fixed paraffin embedded (FFPE). The conditions for analyzing FF tissues using the CODEX platform do not result in effective antibody staining for FFPE tissues. The goal of the research outlined in this grant proposal is to develop conditions and methodologies that enable the analysis of FFPE tissues with the CODEX platform. This will increase the overall applicability of the technology and drastically increase the customer base for Akoya Biosciences Inc. We plan to use the information learned in this Phase SBIR I grant proposal to develop antibody panels validated for FFPE tissues that will be commercialized. This greater development effort will be the focus of a follow-on Phase II SBIR grant application.